Crystallography is an extremely useful tool for scientists, and is therefore a field of research attracting a lot of interest. It is a powerful means that provides precise and detailed description of the three-dimensional structure of the molecules, and is of great help in the understanding of their functions. Crystallography of macromolecules like proteins is extensively used today, academically as well as industrially.
Although three-dimensional structures of simple proteins have been obtained through crystallization methods, it is not always easy to obtain crystals from macromolecules. For example, the preferred conditions for the crystallization of a given molecule can take several hundreds if not thousands of trials. As a result, means and methods have been developed to perform a great number of trials relatively quickly, including hanging-drop and sitting-drop methods. All such methods use the benefit of vapor diffusion to obtain the crystals.
The hanging-drop method is currently the most commonly used technique for scanning various crystallization conditions of macromolecules, such as proteins. It comprises suspending a droplet of approximately 2 to 20 μL of solution containing the macromolecule to be crystallized and a precipitating agent, over a precipitating solution, such as conventional polyethylene glycol 20% or ammonium sulfate 40%, contained in a reservoir or well. The system is then sealed hermetically. After a while, vapor diffusion of the solvent or solvent mixtures between the droplet and the solution in the reservoir reaches equilibrium. The end result is a decrease of water in the droplet, and an increase of the macromolecule and precipitating agent concentration therein, thus causing crystallization of the macromolecule in optimized conditions. The actual technique for the set up of the hanging-drop or sitting-drop experiments is a long and arduous work and has to be performed by qualified and skilled technical personnel.
Conventionally, a commercially available tray made of an inert thermoplastic is material comprising a plurality of reservoirs or wells is prepared, and the precipitating solution is placed in each reservoir or well manually. The macromolecule solution is then mixed with a precipitating agent on a glass plate (coverglass) and the whole is inverted over the wells, thus making the macromolecule solution overhanging the well. Prior to placing a glass plate over a well, the rim of each well is greased to ensure a proper seal. Care must be taken when placing the plate over each well, since the grease can easily contaminate the macromolecule solution. The crystallization process is followed with the help of a microscope. After the crystal is obtained, the glass plate is removed. Again, this must be done with great care to prevent contamination of the crystallized macromolecule with grease, and/or breaking of the glass plate. On top of that, the plates are hardly reusable for any experiment because the grease is hard to remove, and some of it remains on the plates.
An advantage of the hanging-drop and sitting-drop methods is that they provide screening conditions for crystallization, and truly represent a microcrystallization technique. The vapor diffusion in the hanging or sitting drop allows screening of a large range of conditions and necessitates a relatively small amount of macromolecules. Further, it allows a relatively clear visualization of the results, and the eventual crystals are free, i.e., they do not adhere or are stuck to any surface.
Typically, several hundreds of experiments are required to find appropriate crystallizing conditions for the production of high quality crystals. Accordingly, hanging-drop and sitting-drop experiments are a very labor-intensive process demanding skilled and experienced technical personnel. For example, multiple aspirating and dispensing steps of components, multiple greasing steps etc. must be performed in the experimental set up. Further, for each well, a separate coverglass must be manually inverted. The number and complexity of steps can therefore produce an undesirable wide variation in experimental results.
As stated above, grease is conventionally used to provide a seal between the well and the coverglass. Other ways for sealing the system have been proposed. For example, grease can be replaced with immersion oil or an adhesive tape. As with grease, these sealing means have serious drawbacks. Grease is not always easy to dispense around the upper rim of the well, and is a time consuming operation. Technicians repeating the operation thousands of times occasionally suffer physical pain to their hands. Other significant problems and risks are present when manipulating the crystal on a greasy cover slide. The cover slide sometimes breaks during the process, which may cause injury to the technician, in addition to loosing the crystals. The immersion oil is also problematic. One has to use a determined volume of oil. Too much oil leads to contamination within the well, while not enough will lead to non-hermetic seal that may result in the evaporation of the precipitating solution. An adhesive tape allows quicker and simpler manipulations, but all experiments are sealed at the end of the set-up, thus introducing experimental variations between the 1st and the 24th drop. Further, crystals often stick to the tape, rendering impossible the recovery of the crystals, and the operations for the recovery of the drop are also problematic.
These conditions promoted the robotization of the procedure. Some automated crystallization devices already exist. The well-known Cyberlab-200™ apparatus dispenses solutions in wells, greases the upper rim of each well, pours droplets on cover slides held by a vacuum arm, and places the cover slides over the wells. However, such apparatus still has some drawbacks, namely a complicated experimental set-up, and the notable use of grease. Further such apparatus is extremely expensive.
Relevant references in the field include U.S. Pat. No. 2,366,886; U.S. Pat. No. 3,107,204; U.S. Pat. No. 3,297,184; U.S. Pat. No. 3,537,956; U.S. Pat. No. 3,597,326; U.S. Pat. No. 3,649,464; U.S. Pat. No. 3,692,498; U.S. Pat. No. 3,729,382; U.S. Pat. No. 3,745,091; U.S. Pat. No. 3,907,505; U.S. Pat. No. 4,038,149; U.S. Pat. No. 4,154,795; U.S. Pat. No. 4,495,289; U.S. Pat. No. 4,917,707; U.S. Pat. No. 5,271,795;
It would therefore be highly desirable to develop a device for crystallizing macromolecules that would overcome the above deficiencies. Such device would eliminate the requirement of external means like grease, oil or an adhesive tape to seal the well and the cover, and would preferably be easy to manipulate, either manually or automatically. Ultimately, the process of experimental set up of the device would be greatly facilitated and accelerated, while simultaneously eliminating possible risks of contamination of the eventual crystals. Finally, such device should be usable for various crystallization processes such as hanging-drop or sitting-drop processes.